Pacific oyster Crassostrea gigas exposed to prolongedthermal stress Anne-Leila Meistertzheim1, Arnaud Tanguy2, Dario Moraga1and Marie-The´re`se The´bault1 1 Laboratoire des Sciences de l
Trang 1Pacific oyster Crassostrea gigas exposed to prolonged
thermal stress
Anne-Leila Meistertzheim1, Arnaud Tanguy2, Dario Moraga1and Marie-The´re`se The´bault1
1 Laboratoire des Sciences de l’Environnement Marin, Institut Universitaire Europe´an de la Mer, Universite´ de Bretagne occidentale, Plouzane´, France
2 Laboratoire Adaptation et Diversite´ en Milieu Marin, Station Biologique, Roscoff, France
The fluctuating thermal nature of the marine
environ-ment induces physiological changes in ectotherms that
require molecular and gene expression adjustments [1]
Comparative gene expression studies can be used to
characterize these adjustments and lead to a better
understanding of organismal responses to
environmen-tal change Gene expression datasets can be clustered
into groups of genes that represent different
compart-ments of cellular function, and changes in the
expres-sion of genes from these clusters can be used to
formulate hypotheses as to how different tissues and
whole organisms respond to particular biotic or abiotic
stresses Few studies have addressed changes in gene
expression in response to temperature variation on
marine organisms Alterations in gene expression have
been observed in fish acclimated to constant tempera-tures and then exposed to daily temperature fluctua-tions [2] or to a strong heat stress [3] However, few molecular investigations have focused on the thermal stress response in marine invertebrates [4,5], particu-larly in the context of global changes and the potential effects on marine invertebrates [6,7]
The Pacific oyster Crassostrea gigas is a eurythermic bivalve mollusc that colonizes most of the western coast of Europe This species prefers sheltered estua-rine waters, where it is found in intertidal and shallow subtidal zones Within their geographic range, oysters typically experience and respond to seasonal tempera-tures ranging from 4 to 24C [8] In the coldest regions inhabited by C gigas, such as Brittany,
Keywords
climate; Crassostrea gigas; gene expression;
heat stress; prolonged thermal stress
Correspondence
M T The´bault, Laboratoire des Sciences
de l’Environnement Marin, UMR-CNRS
6539, Institut Universitaire Europe´en de la
Mer, Universite´ de Bretagne Occidentale,
Place Nicolas Copernic, 29280 Plouzane´,
France
Fax: +33 2 98 49 86 45
Tel: +33 2 98 49 86 12
E-mail: marie-therese.thebault@univ-brest.fr
(Received 5 April 2007, revised 17 October
2007, accepted 19 October 2007)
doi:10.1111/j.1742-4658.2007.06156.x
Groups of oysters (Crassostrea gigas) were exposed to 25C for 24 days (controls to 13C) to explore the biochemical and molecular pathways affected by prolonged thermal stress This temperature is 4C above the summer seawater temperature encountered in western Brittany, France where the animals were collected Suppression subtractive hybridization was used to identify specific up- and downregulated genes in gill and mantle tissues after 7–10 and 24 days of exposure The resulting libraries contain 858 different sequences that potentially represent highly expressed genes in thermally stressed oysters Expression of 17 genes identified in these libraries was studied using real-time PCR in gills and mantle at differ-ent time points over the course of the thermal stress Differdiffer-ential gene expression levels were much higher in gills than in the mantle, showing that gills are more sensitive to thermal stress Expression of most transcripts (mainly heat shock proteins and genes involved in cellular homeostasis) showed a high and rapid increase at 3–7 days of exposure, followed by a decrease at 14 days, and a second, less-pronounced increase at 17–24 days
A slow-down in protein synthesis occurred after 24 days of thermal stress
Abbreviations
CTSL, cathepsin L; EST, expressed sequence tag; HYPK, Huntingtin-interacting protein K; HSP, heat shock protein; LDH, lactate
dehydrogenase; MTA-1, metastasis-associated protein 1; SSH, suppression substractive hybridization.
Trang 2France, summer water temperatures only occasionally
reach 21C for short periods of up to a few days
Recent studies on C gigas suggest that complex
inter-actions between temperature and food quality and
quantity affect gametogenesis [9,10] Reproductive
development is temperature dependent in C gigas and
typically occurs between February and September
along the western Atlantic coast of France [11]
Spawning occurs at a minimum seawater temperature
of 19 C [12,13] One study focused on the
expres-sion of heat shock proteins (HSPs) in C gigas, at the
molecular and physiological level, in response to heat
stress throughout the year [7]
The aim of this study was to improve our knowledge
of differentially expressed genes in C gigas exposed to
a temperature slightly above the upper natural water
temperature in the area Our study is the first to apply
an overall genomic approach to the study of the
response of C gigas to prolonged heat stress We used
animals outside the season of reproductive
develop-ment and spawning in order to measure temperature
stress without the onset of reproduction Using
sup-pression substractive hybridization (SSH), we identified
genes that were up- and downregulated 7–10 and
24 days after transfer from 13 to 25C Subsequently,
genes likely to be associated with thermal stress were
quantified using quantitative real-time PCR
Results
Suppression subtractive hybridization
SSH libraries were constructed from pooled gills and
mantle of C gigas after 7–10 and 24 days of exposure
to different temperature treatments The search for
homology using the blastx program revealed 858
different sequences, of which 536 ( 62%) remain
unidentified Expressed sequence tags (ESTs) similar to
genes potentially involved in a thermal response were
subsequently clustered into 15 distinct functional
cate-gories: cell differentiation (including cell migration,
adhesion, proliferation and apoptosis), cellular
com-munication (including signal transduction), cellular
stress (including inflammation and immune response),
cytoskeleton and cell structure (including cellular
matrix and cellular trafficking), detoxification,
ener-getic metabolism, lipid metabolism, receptors and
channels, regulation of nucleosides, nucleotides and
acid nucleic metabolism, reproduction, respiratory
chain, transcriptional processing, translational and
post-translational processing, general metabolism
and other functions and ribosomal proteins (Fig 1
and supplementary Tables S1–S4) Among the 322
recognized protein-coding genes, 191 new sequences were obtained in C gigas and 131 had been identified previously, of which 88 genes encode ribosomal pro-teins in both forward and reverse libraries Among the newly known sequences, only one corresponded to a gene specific for mantle (mantle gene 4) No cellular signalling genes were identified in samples taken after
24 days Cytoskeletal genes, translation and ribosomal genes were less abundant on warming Only respira-tory genes were more abundant on warming
Gene expression patterns from different functional categories during temperature acclimation
Using real-time PCR we conducted a time-course study to compare transcript expression in oysters exposed to thermal stress at 25C relative to control animals maintained at 13C Seventeen transcripts analysed after 0, 3, 7, 14, 17 and 24 days of exposure (Fig 2) belonged to categories previously implicated in the stress response: (a) cell proliferation and differenti-ation: metastasis-associated protein 1 (MTA-1), Hun-tingtin-interacting protein K (HYPK), cystatin B, cathepsin L (CTSL, EC 3.4.22.15) (these proteins are known tumor markers) [14,15], QM protein (transcrip-tional control of cell differentiation and proliferation) [16,17], Ras family GTP-binding protein Rho1p (dif-ferentiation); (b) cellular stress: HSP70, HSP70 kDa protein 12A, HSP23, chaperonin-containing TCP1 (alternative name CCT) subunit 7, isoform b (chaper-ones), inhibitor of kappa light polypeptide (inflamma-tion); (c) antioxidant defense: non-selenium glutathione peroxidase (EC 1.11.1.7); (d) metabolism of nitrogen and ammonia detoxification: glutamine synthetase (EC 1.4.1.13); (e) membrane fluidity: D9 desaturase (EC 1.14.19.1); (f) energetic metabolism: d-lactate dehydrogenase (d-LDH, EC 1.1.1.28, anaerobic metab-olism), citrate synthase (EC 2.3.3.1, aerobic metabo-lism); and (g) translational processing (translation initiation factor eIF-2B delta subunit) Normalized expression data are summarized in Table 1
In the gills, all transcripts selected in the forward SSH library at 25C, except citrate synthase, showed
an initial expression peak at days 3–7, followed by a decrease at day 14, and then a smaller increase at days 17–24 at 25C compared with controls (13 C) (Fig 2A and Table 1) The most differentially expressed transcripts at 25C were HSPs, MTA-1 pro-tein, chaperonin-containing TCP1 subunit 7, isoform b and d-LDH Gene expression was less pronounced in mantle relative to the gills (Fig 2) In the mantle, some transcripts (HSP70, HSP23, MTA-1 protein, Rho1p,
Trang 3D9-desaturase and glutamine synthetase) showed a
peak of overexpression at days 14 and⁄ or 17 In
con-trast to observations on gill tissue, mantle levels of
HSP12A, MTA-1 protein, Rho1p, D9-desaturase and
citrate synthase transcripts were significantly lower at
25C on day 3 Variation in the expression of d-LDH
and chaperonin-containing TCP1 subunit 7, isoform b
genes was not significant
Different profiles were observed for transcripts
selected from the reverse SSH library at 13C (Fig 2B
and Table 1) In gill tissue, inhibitor of kappa light
polypeptide, non-selenium glutathione peroxidase,
CTSL and translation initiation factor eIF-2B were
highly expressed at days 3 and⁄ or 7 at 25 C relative
to control (13C) CTSL, non-selenium glutathione
peroxidase and translation initiation factor eIF-2B
were downregulated at day 24 in gills at 25C
Expres-sion of the HYPK gene did not show much variation,
although it increased significantly from the beginning
to the end of the experiment Cystatin B, inhibitor of
kappa light polypeptide and QM protein (60S ribo-somal protein) showed a biphasic expression pattern
In mantle cells, variation in expression levels of CTSL and inhibitor of kappa light polypeptide genes was not significant Overexpression of cystatin B, non-selenium glutathione peroxidase and translation initiation factor eIF-2B transcripts was limited to a relatively short time-window at days 14 and⁄ or 17 HYPK and QM protein were underexpressed at days 3, 7 and 24 and day 7, respectively
Discussion
Of 322 genes identified in this study, 191 partial sequences had not been identified previously in
C gigas Of the 131 known genes, 88 encode ribo-somal proteins and had been identified previously in
C gigas responding to environmental stresses such as hydrocarbons, pesticides and hypoxia [18–20] Thus, their gene products appear to be important for
Cell differentiation, migration, adhesion, proliferation, apoptosis
Cellular communication, signal transduction Cellular stress, inflammation, immune function Cytoskeleton, structure, matrix and cellular trafficking
Detoxification Energetic metabolism General metabolism, others functions Lipid metabolism
Receptors and channel Regulation of nucleoside, nucleotide and nucleic acid metabolism
Reproduction Respiratory chain Transcriptional processing Translational and post-translational processing
Ribosomal proteins
3%
10%
9%
1%
5%
6%
2%
9%
4%
1%
11%
5%
6%
26%
11%
8%
6%
12%
3%
9%
2%
9%
3%
35%
2%
5%
5%
15%
1% 7%
5%
8%
6%
17%
32%
6%
8%
11%
3%
3%
14%
17%
28%
8%
5%
3%
Fig 1 Functional classification of the sequences identified in SSH libraries which matched known genes corresponding to the 100% value SSH were made from pooled gills and mantle of C gigas Genes were clustered into 15 categories according to their putative biological function A1 and A2, 25 and 13 C at 7–10 days; B1 and B2, 25 and 13 C at 24 days.
Trang 4Relative expression
Metastasis associated protei
Chaperonin containing T
D-lactate dehy
kappa light poly
Non-selenium g
T fa
Trang 5metabolic adjustments during stress in general
How-ever, the expression patterns observed were tissue
spe-cific with gills being more responsive than mantle We
hypothesize that the observed patterns reflect
func-tional differences between these two tissues A number
of genes that were highly expressed in gills showed
a biphasic expression pattern, consisting of a strong
short- and a moderate long-term response Moreover, after 7–10 days of exposure, we detected differential expression of a number of genes that encode elements
of the transcription and translation machinery, includ-ing transcription factors, ribosomal proteins and elon-gation factors After 24 days of exposure to elevated temperature, the differential expression profile was
Table 1 Expression patterns in gills (G) and mantle (M) throughout the experiment at 25 versus 13 C For each gene, + (or –) represents significant relative upregulation (or downregulation): + ⁄ ) from 1.2- to 2-fold; ++ ⁄ )) from 2- to 5-fold; +++ ⁄ )) from 5- to 10-fold; ++++
> 10-fold NS, not significant *P < 0.05, **P < 0.01, ***P < 0.001.
Days of exposure Tissue
Cell proliferation and differentiation
**
* NS
Cellular stress
Chaperonin containing TCP1,
subunit 7, isoform b, isoform 1
Inhibitor of kappa light polypeptide
enhancer in B cells, kinase complex
Antioxidant defeNSe
Metabolism of nitrogen and ammonia detoxification
Membrane fluidity
Energetic metabolism
Translational processing
Translation initiation factor
eIF-2B delta subunit
Trang 6dominated by strong downregulation of genes involved
in protein synthesis, such as the translation initiation
factor eIF-2B delta subunit, suggesting a slowing of
protein synthesis These findings may suggest that
transcription factors are regulated though a feedback
mechanism, inducing their own inactivation [21]
Changes in gene expression of organisms subjected to
thermal stress are known to involve major adjustments
in the expression of ribosomal genes and genes coding
for proteins involved in RNA metabolism and protein
synthesis In fact, protein synthesis in marine snails
was inactivated at temperatures approaching lethal
values [22]
Under mild thermal stress at 25C, genes coding for
antistress proteins were differentially expressed
Protec-tion against cellular stress, inflammaProtec-tion and
stimula-tion of immune funcstimula-tion appear to be important
components of responses to thermal stress HSPs play
an essential role in maintaining protein homeostasis
during exposure to proteotoxic stressors [23] They
function by interacting with stress-denatured proteins
and preventing their aggregation and⁄ or degradation
[24] HSP induction may therefore have an adaptative
value for organisms facing thermal stress and
signifi-cant ecological and biogeographical implications for
species distribution and their thermotolerance limits
[22,25] Tissue-specific de novo HSPs synthesis was
induced in C gigas following exposure to 25C, which
is 4C higher than the highest sea surface
tempera-tures recorded in its distribution range Two HSPs
(HSP70 and HSP23) were greatly and rapidly
upregu-lated in gills but slightly less and later in mantle One
inducible and two constitutive isoforms of HSP70 are
synthesized in the gills and mantle of C gigas [26]
The expression level of the constitutive forms increases
after thermal stress, whereas the inducible one is
expressed only after exposure to 32C [7] These
results suggest that the overexpression of HSP70 we
observed might correspond to the constitutive form
HSP23 is a small heat shock protein highly induced
following stress Small HSPs are differentially
expressed between tissues and through the different
stages of development [27] A third HSP, HSP12A
(alternative name 150 kDa oxygen-regulated protein;
ORP150), was induced in gill tissue only This
chaper-one, located in the endoplasmic reticulum, plays an
important role in maintaining cell viability in response
to stress [28] Among other chaperones, the
chapero-nin-containing TCP1 (subunit 7, isoform b) presented
the same expression pattern in response to heat stress
in our study This complex, involved in folding actin,
tubulin and cyclin E, among other proteins [29], is also
upregulated in response to chemical stress [30] Hence
TCP1 may play an important role in the recovery of cells after protein damage, by assisting the folding of cytoskeletal proteins that are actively synthesized and⁄ or renatured under these conditions The upregu-lation of all of these chaperones confirms the severity
of the thermal stress under our experimental condi-tions
A number of genes encoding structural components
of the cytoskeleton and proteins involved in contractile functions (including actin, tubulin myosin and profilin) were differentially expressed in C gigas in response to prolonged heat stress, some were induced and some repressed Rho1p, for example, encodes for a protein involved in numerous processes including actin fila-ment organization and is expressed in response to envi-ronmental changes [31] In this study, Rholp was rapidly upregulated in gills and later in mantle during warming These results suggest that extensive cytoskel-etal reorganization occurs in response to heat stress, as reported for fish gills [3]
Furthermore, several genes associated with the regu-lation of cell homeostasis were differentially expressed
in our study Some genes were differentially expressed
in both tissues, showing increased apoptotic⁄ autopha-gic activity Among these genes, MTA-1 was strongly expressed in gills during warming, whereas it was ini-tially downregulated in mantle CTSL, a highly potent endoprotease involved in lysosomal bulk proteolysis, was strongly expressed, but only in gills The upregula-tion of CTSL, combined with the downregulaupregula-tion of its reversible binding inhibitor cystatin B, implies that active protein degradation was taking place in the gills upon warming to 25 C Two less well-known genes, putatively involved in proliferation and apoptosis, QM protein and HYPK were both differentially expressed between the tissues QM protein, also known as ribo-somal protein L10, is a transcription cofactor that inhibits activation of AP-1 transcription factors QM protein is implicated in the conversion of a broad vari-ety of extracellular signals generated by growth factors, tumour promoters or genotoxic drugs [16,32] In the sponge, Suberites domuncula, QM protein expression was significantly higher in tissues undergoing induced apopotosis [33] On warming to 25C, upregulation of
QM protein occurred rapidly in gills and later in man-tle HYPK, identified as a antiapoptotic protein [34], displayed the same expression pattern as QM protein
in both tissues These results suggest that, in C gigas, these proteins are induced to prevent pathologies such
as inflammation and tumorigenesis during prolonged thermal stress
The cellular stress response has an energetic cost and control of the balance between ATP supply and
Trang 7demand in the ciliated gill may become altered during
thermal stress In C gigas, stressors such as
hydro-carbons, herbicides, parasite infection or hypoxia
[18–20,35], affect the expression of genes involved in
energetic metabolism and our results show that
changes in transcript levels of a number of genes
involved in metabolic regulation also occur in response
to temperature In gill tissue, prolonged heat stress
resulted in the rapid induction of several
ATP-generat-ing enzymes includATP-generat-ing the tricarboxylic acid cycle
citrate synthase, suggesting that there was a need for
rapid aerobic ATP production In the early phase of
warming, the rapid induction of LDH in gills may
indicate that anaerobic metabolism is required The
LDH that we identified was d-specific Many
system-atic studies have shown that d- or l-specific LDHs are
present in all invertebrate groups [36] We also
observed that the glutamine synthetase gene was
up-regulated in both tissues, as previously observed in
response to hydrocarbons, herbicides or hypoxia
[18–20] In vertebrates, glutamine synthetase occupies a
central position in nitrogen metabolism and is linked
to amino acid turnover, nitrogen detoxification,
nucleotide biosynthesis and more generally to growth
[37] Although the capacity for glutamine biosynthesis
is generally weak or absent in molluscs [38], a recent
study reported the accumulation of glutamine
associ-ated with an upregulation of glutamine synthetase in a
bivalve species in response to aerial exposure [39]
Glutamine synthesis may also be an ammonia
detoxifi-cation mechanism in invertebrates
Genes involved in fatty acid metabolism are
expected to be affected by temperature Among these,
D9 desaturase has been extensively studied in
numer-ous animal groups including mammals, chicken, fish
and insects [40,41] High temperatures typically
increase membrane fluidity in temperate eurytherms
[42] The upregulation of D9 desaturase that we
observed in gills, and later in mantle, agrees with this
pattern A similar pattern was previously observed in
C gigas in response to experimental hypoxia,
suggest-ing that the regulation of this enzyme may be affected
primarily by oxidative stress [24] Recent studies on
intertidal bivalves show that critical warming may
exacerbate cellular oxidative stress [43] In many
spe-cies, the increase in lipid peroxidation and reactive
oxygen species concentration in cells following heat
stress has already been shown to modify the activity of
antioxidant enzymes such as GPx [44] Non-selenium
glutathione peroxidase is involved in detoxification by
reducing fatty acid hydroxyperoxides and H2O2[45] In
our study, the level of the non-selenium glutathione
peroxidase transcript appears strongly upregulated in
gills during the first week of thermal exposure A simi-lar thermal stress response, associated with oxidative stress, has also been observed in other marine poikilo-thermic species including molluscs [43,46–48]
Our results represent the first stages of investigation into the molecular response of oysters to high tempera-tures, focusing on early winter, outside the gametogen-esis period Future efforts will focus on the search for functional polymorphism in some of the genes poten-tially regulated by temperature in oyster populations located at the limits of the species distribution area
Experimental procedures Thermal acclimation and experimental design
Adult oysters (length 85 ± 5 mm) were collected from La Pointe du Chaˆteau (Brittany, France) in November 2004
at ambient temperature ( 13 C) in aerated 0.22-lm fil-tered seawater tanks for 21 days Groups of oysters were then exposed to two laboratory-controlled temperature regimes in 40 L tanks: 60 oysters were acclimated for
encountered in summer in southern Brittany), and a control
Oysters were fed three times a week with a microalgal sus-pension (containing Isochrysis galbana and Pavlova lutheri)
No oysters died during the experiment
For each of the experimental conditions, oysters were sampled at 0, 3, 7, 10, 14, 17 and 24 days following the start of the treatments Gill and mantle tissues were dis-sected, rapidly frozen in liquid nitrogen and stored at )80 C until analysis Pools of gill and mantle were pre-pared on these sampling dates by taking 50 mg of each tissue from each of 10 individuals
RNA extraction
Total RNA was extracted using TRIzol Reagent
experiments, polyadenylated RNA was isolated using the PolyATtractmRNA Isolation System (Promega, Madison, WI) according to the manufacturer’s instructions RNAs were resuspended in RNase-free water and their quantity was assessed by spectrophotometry
Suppression subtractive hybridization
Messenger RNA was extracted from mantle and gills of
Two micrograms of mRNA (1 lg from the gills and 1 lg from the mantle) were used as the template for SSH
Trang 8(Clontech, Palo Alto, CA) Hybridization and subtraction
steps were carried out in both directions For forward
subtraction Four libraries (two forward and two reverse)
were thus constructed PCR products were then purified
and cloned into pGEM-T vector (Promega) Five hundred
white colonies per library were grown on Luria–Bertani
ran-domly selected clones were single-pass sequenced using an
ABI 3730 sequencer with the sequencing kit ABI Big dye
terminator version 3.1 at the Genoscope Sequencing Center
(Evry, France) Sequences were then analyzed using BlastX
algorithm available from the National Center for
Biotech-nology Information (NCBI) and the EST sequences were
then submitted to its dbEST and GenBank databases (see
supplementary Tables S1–S4)
Real-time PCR analyses
Real-time PCR was used to analyse the expression profiles
of some selected genes involved in cell proliferation and dif-ferentiation, cellular stress, antioxidant defence, metabolism
of nitrogen and ammonia detoxification, membrane fluidity, energetic metabolism and translational processing Total RNA was extracted from gills and mantle of 10 oysters
A pool of the 10 RNA samples was made for each tissue at each sampling point in a proportional manner according to the amount of total RNA collected from each animal Reverse transcription was performed on 20 lg RNA from each pool using the oligo(dT) anchor primer (5¢-GAC
murine leukaemia virus (M-MLV) reverse transcriptase (Promega) Real-time PCR was performed in triplicate with
Table 2 Combinations of primers used in real-time PCR expression analysis.
GCTTGGCTACTGGACCATCAA
CAGTTCCTCGGGCCAACA
CATCTTCGGCCGTCTTTCC
TGGATCGCCAAAAACTCATG
GCTTGGCTACTGGACCATCAA
GCGCAACTAATGCTTCCACAA
AATCAGACGGCCGGTATGTG
CTCATCCTCCACCGGATTGT
ACCAGAAGACATTACAGTGAAAATTGA
CGTCCACTGAGAGGATGAGACA Inhibitor of kappa light polypeptide enhancer in B cells, kinase
complex-associated protein
AAAGCAGAGCAGAAAAAGTGGAA GGACAATGCCGCGATCAG
GGGATGGAGGGTAAGACCATACA
GCTGGCACCACGATTGG
CCGACCATGTGGCGTTTAGT
TTCGTCGGACACAGAGTCTCCCAATTCTC
CGCCATATTGCTTGACAGCTACT
CACTTTAGTAGCCTCTTGCATTGC
TGCTCAATCTCGTGTGGCTAAACGCAACTTG
Trang 95 lL cDNA (1⁄ 20 dilution) in a total volume of 20 lL,
using a 7300 Real-Time PCR System (Applied Biosystems,
Foster City, CA) The concentrations of the reaction
components were as follows: 1· Absolute QPCR SYBR
Green ROX Mix (ABgene, Epsom, UK) and 70 nm of
each primer Oligonucleotide primer sequences used to
amplify specific gene products are shown in Table 2
Reactions were realized with activation of Thermo-Start
ampli-fication of the target cDNA (45 cycles of denaturation at
each amplification plate), a negative control
(nonreverse-transcribed total RNA) and blank controls (water) for
each primer pair PCR products were then purified,
cloned and sequenced for confirmation
For gene expression calculation, the threshold value
(Ct) was determined for each target as the number of
cycles at which the fluorescence rose appreciably above
the background fluorescence PCR efficiency (E) was
cal-culated for each primer pair by determining the slope of
standard curves obtained from serial dilution analysis of
cDNA from different experimental samples (treatment and
control), using the method described by Yuan et al.[49]
Individual real-time PCR efficiencies (E) for target or
Results are presented here as changes in relative
expres-sion normalized to the reference gene (ribosomal 18S),
using the method described by Pfaffl [50] and determined
using the equation:
Statistical analysis
The variations in gene expression were analyzed with
statistical analyses were performed using the triplicate
real-time PCR assay values obtained for each sample; the graphs
(Fig 2) present the mean values with standard deviations
Acknowledgements
This research program was financially supported by
the national program PROGIG (Prolife´ration de
Crassostrea gigas, LITEAU II) and the PolyGIGAS
program of the Bureau des Ressources Ge´ne´tiques (n05 ⁄ 5210460 ⁄ YF) The authors are grateful to Helen McCombie and Carolyn Friedman for English correc-tions
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